1. Field of the Invention
Enzyme immunoassays are a very important tool in diagnostics and have several advantages, particularly over radioimmunoassays including, for example, the avoidance of radiation hazards, the convenience of detecting a chromogenic response, the avoidance of reagent instability due to radioactive decay, and the opportunity to amplify the response due to the ability of enzymes to generate many molecules of product per molecule of enzyme.
Enzyme immunoassays operate upon the principle that enzymes are biological catalysts that accelerate specific chemical reactions. Since a single molecule of catalyst can transform many molecules of substrate to product by repeating the catalytic reaction, the catalyst acts as an amplifier. Enzymes are therefore easily detectable at very low concentrations. This sensitivity makes enzymes useful as immunochemical labels. In an enzyme immunoassay for an analyte in a sample, an appropriate enzyme that is conjugated to one of the immunoreactants becomes bound to another immunoreactant or to the analyte and the activity of the enzyme is determined by measuring the conversion of an enzyme substrate to a product. The amount of product is an indication of the amount of analyte in the sample.
In heterogeneous enzyme immunoassays such as an ELISA assay, the unbound enzyme is first separated from the bound enzyme before measuring the enzyme activity.
Other assays, known as homogeneous enzyme immunoassays, detect the amount of enzyme activity as an indication of the amount of analyte, without the need to separate unbound enzyme from enzyme that is bound to an immunoreactant. These assays use an enzyme whose activity is capable of being modulated as a result of binding to the immunoreactant. One such method uses an enzyme-labeled ligand and a receptor, where the enzyme activity changes when the receptor is bound to an analyte instead of to the enzyme. Another such method involves using an enzyme-labeled receptor and measuring a change in enzyme activity upon binding of the receptor to a ligand. Another method, known as an enzyme channeling immunoassay, depends on a change in the enzyme activity when two enzymes are caused to come into close proximity with each other as a result of immunochemical binding. The enzymes are related in that the product of one enzyme is a substrate for the other. An excellent overview of enzyme immunoassays is presented in "Enzyme-Immunoassay", Edward T. Maggio, ed., CRC Press, Inc. (1980).
In homogeneous enzyme immunoassay methods, it is often difficult to adequately modulate the activity of an enzyme reagent with an antibody and some enzymes provide little or no modulation. The present invention provides an improvement in homogenous enzyme immunoassays wherein it is possible to modulate the activity of enzymes that heretofore have not been useful in enzyme immunoassays and thereby increase the versatility and sensitivity of the homogeneous enzyme immunoassay method.
2. Description of the Related Art
Anti-enzyme antibodies have been described as enzyme activity modulators. In Ngo, et al., FEBS Letters 116(2):285-288 (1980), an enzyme modulator is covalently linked to a ligand that is similar to the analyte such that the amount of modulator free to regulate enzyme activity is dependent upon the amount of analyte present. For example, ligand that has been labeled with anti-horseradish peroxidase (HRP) antibodies competes with analyte for binding to anti-analyte antibodies. Upon addition of HRP, any ligand-labeled anti-HRP antibodies remaining become bound to HRP, making it enzymatically inactive.
In U.S. Pat. No. 4,686,181, anti-enzyme antibodies are used to inhibit the activity of glucose-6-phosphate dehydrogenase (G6PDH). Anti-G6PDH is conjugated to the analyte or an analog of the analyte. A liquid medium containing the analyte is combined with a binding agent for the analyte, the anti-G6PDH-conjugate, and G6PDH. G6PDH activity is then measured. Skold, et al., Journal of Immunology 138(10):3408-3414 (1987) also describes use of anti-G6PDH antibodies to modulate G6PDH activity.
IgG antibodies have also been described as enzyme activity modulators. Wei, et al., Clin. Chem. 23(8): 1386-1388 (1977) describes inhibition of enzymatic activity by IgG. When IgG binds to anti-IgG antibodies labeled with phospholipase C, the phospholipase C activity is suppressed. The catalytic site of the enzyme is presumably masked, which prevents its interaction with substrate.
Hybrid antibodies have been described as enzyme modulators. In Ashihara, et al., Journal of Clinical Laboratory Analysis 1:77-79 (1987), hybrid antibodies are described that are capable of binding to antigen or enzyme competitively and of inhibiting enzyme activity.
Chemical compounds have also been used to modify enzyme activity. United Kingdom Patent No. 1,595,101 describes an enzyme modifier immunoassay using a modifier, which is bound to a ligand or to a receptor for the ligand. The assay involves forming a complex between the modifier labeled member and the analyte, followed by addition of enzyme and substrate to the assay mixture, and measurement of enzyme activity. Miyake, et al., Agric. Biol. Chem. 52(7):1649-1654 (1988) describes several compounds capable of inhibiting the enzyme activity of .beta.-galactosidase.
Inhibitors, either anti-enzyme antibodies or chemical compounds, have been used to reduce enzyme activity. EPO No. 0,272,691 describes an assay where antibody or antigen bound to a solid phase is combined with a sample to be assayed and an enzyme-labeled antibody or antigen. An insoluble solid carrier bound to an enzyme inhibitor is added to the liquid remaining after the antigen-antibody interaction, to reduce the activity of the enzyme in the unreacted enzyme-labeled antibody or antigen present in the liquid.